title: Protein expression, biochemical pharmacology of signal transduction, and relation to intraocular pressure modulation by bradykinin B₂ receptors in ciliary muscle.
abstract: OBJECTIVE
  To examine the bradykinin (BK) B₂-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied.
  
  
  METHODS
  Previously published procedures were used throughout these studies.
  
  
  RESULTS
  The human and monkey ciliary bodies expressed high levels of B₂-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca²⁺](i) mobilization in primary h-CM cells (BK EC₅₀=2.4±0.2 nM > Hyp³,β-(2-thienyl)-Ala⁵,Tyr(Me)⁸-(®)-Arg⁹-BK (RMP-7) > Des-Arg⁹-BK EC₅₀=4.2 µM [n=3-6]), and this was blocked by B₂-selective antagonists, HOE-140 (IC₅₀=1.4±0.1 nM) and WIN-63448 (IC₅₀=174 nM). A phospholipase C inhibitor (U73122; 10-30 µM) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca²⁺](i) mobilization. Total prostaglandin (primarily PGE₂) secretion stimulated by BK and other kinins in h-CM cells was attenuated by the cyclooxygenase inhibitors bromfenac and flurbiprofen, and by the B₂-antagonists. BK and RMP-7 (100 nM) induced a twofold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and BK (0.1-1 µM; at 24 h) caused a 1.4-3.1-fold increase in promatrix metalloproteinases-1-3 release. Topical ocular BK (100 µg) failed to alter IOP in cynomolgus monkeys. However, intravitreal injection of 50 µg of BK, but not Des-Arg⁹-BK, lowered IOP in rabbit eyes (22.9±7.3% and 37.0±5.6% at 5 h and 8 h post-injection; n=7-10).
  
  
  CONCLUSIONS
  These studies have provided evidence of a functional endogenously expressed B₂-receptor system in the CM that appears to be involved in modulating IOP.
authors.affiliation: Pharmaceutical Research, Alcon Research, Ltd., Fort Worth, TX, USA. naj.sharif@alcon.com
authors.forename: Najam A
authors.initials: NA
authors.lastname: Sharif
authors.forename: Shouxi
authors.initials: S
authors.lastname: Xu
authors.forename: Linya
authors.initials: L
authors.lastname: Li
authors.forename: Parvaneh
authors.initials: P
authors.lastname: Katoli
authors.forename: Curtis R
authors.initials: CR
authors.lastname: Kelly
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Shutong
authors.initials: S
authors.lastname: Cao
authors.forename: Rajkumar
authors.initials: R
authors.lastname: Patil
authors.forename: Shahid
authors.initials: S
authors.lastname: Husain
authors.forename: Laura
authors.initials: L
authors.lastname: Klekar
authors.forename: Daniel
authors.initials: D
authors.lastname: Scott
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001920:Bradykinin / Q000008:administration & dosage / Q000494:pharmacology; D065094:Bradykinin B2 Receptor Antagonists; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007429:Intraocular Pressure / Q000187:drug effects / Q000502:physiology*; D008252:Macaca fascicularis; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011453:Prostaglandins / Q000378:metabolism; D011817:Rabbits; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors
chemical_list: D065094:Bradykinin B2 Receptor Antagonists; D016861:Cyclooxygenase Inhibitors; D010455:Peptides; D011453:Prostaglandins; D043782:Receptor, Bradykinin B2; D009569:Nitric Oxide; D048049:Extracellular Signal-Regulated MAP Kinases; D020782:Matrix Metalloproteinases; D001920:Bradykinin